High intensity sweeteners possess a sweetness level many times exceeding that of sucrose. They are widely used in diet and calorie-reduced food as being essentially non-caloric, not affecting blood glucose level, and provide little or no nutritive value. In contrast, natural caloric sweeteners such as sucrose, fructose, and glucose are not suitable for diet and calorie-reduced food for being highly caloric, even though they provide the most desirable taste to consumers.
Stevia rebaudiana Bertoni is a perennial shrub of the Asteraceae (Compositae) family native to certain regions of South America. The leaves of the plant contain from 10 to 20% of diterpene glycosides, which are around 150 to 450 times sweeter than sugar. The leaves have been traditionally used for hundreds of years in Paraguay and Brazil to sweeten local teas and medicines.
At present there are more than 230 Stevia species with significant sweetening properties. The plant has been successfully grown under a wide range of conditions from its native subtropics to the cold northern latitudes.
The extract of Stevia rebaudiana plant contains a mixture of different sweet diterpene glycosides, which have a single base—steviol and differ by the presence of carbohydrate residues at positions C13 and C19. These glycosides accumulate in Stevia leaves and compose approximately 10%-20% of the total dry weight. The steviol glycosides include Dulcoside A, Rebaudiosides A, B, C, D, E, and F, steviolbioside, and Rubusoside. Typically, on a dry weight basis, the four major glycosides found in the leaves of Stevia are Dulcoside A (0.3%), Rebaudioside C (0.6-1.0%), Rebaudioside A (3.8%) and Stevioside (9.1%). Among steviol glycosides only Stevioside and Rebaudioside A are available in commercial scale.
Steviol glycosides have zero calories and can be used wherever sugar is used. They are ideal for diabetic and low calorie diets. In addition, the sweet steviol glycosides possess functional and sensory properties superior to those of many high potency sweeteners.
Rebaudioside D (CAS No: 63279-13-0) is one of the sweet glycosides found in Stevia rebaudiana. 
Studies show that highly purified forms of Rebaudioside D possess very desirable taste profile, almost lacking bitterness, lingering licorice aftertaste typical for other Steviol glycosides. These properties multiply the significance of Rebaudioside D and attract great interest for methods of preparation of highly purified forms of Rebaudioside D.
It has to be noted that in commercially available Stevia rebaudiana varieties rebaudisoide D content is very low. Generally on a dry weight basis the leaves of Stevia rebaudiana contain 0-0.1% rebaudioside D. Therefore there's a need of developing new varieties of Stevia rebaudiana with higher content of rebaudioside D. One of the key conditions for such development work is the availability of simple and high throughput testing methodologies, which can be applied on a large number of samples.
On the other hand, the testing methodologies which exist today for steviol glycosides' analysis employ very sophisticated and expensive HPLC techniques with very low throughput. These methods, although robust and well established, are time consuming, expensive, require specialised technicians and instrumentation, and the number of samples that can be processed daily is small. In addition, the amounts of chemicals and toxic solvents that are used often have a high environmental risk.
These disadvantages clearly show the need for developing fast, easy-to-use, robust, sensitive and cost-effective techniques for high throughput analysis of steviol glycosides, particularly rebaudioside D in various matrices, including plant biomass.